NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.

The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated by proteolytic cleavage of its subunits by channel activating proteases (CAPs). Dysregulation of ENaC leads to increased sodium absorption which contributes to airways dehydration and thickened mucus. Impaired mucociliary clearance (MCC), characteristic of cystic fibrosis (CF) lung disease, predisposes the individual to chronic cycles of infection and inflammation, irreversible lung damage and pulmonary decline. Inhibition of CAPs therefore presents a promising target for therapy as airway rehydration through reduced ENaC activation, has the potential to restore MCC. Previous work by Reihill et al has shown that QUB-TL1, an active site-directed protease inhibitor, effectively inhibits trypsin-like enzymes and reduces ENaC-mediated sodium absorption (1). We have developed a number of other novel compounds therefore the aim of this study was to assess their ability to inhibit CAPs and to investigate downstream effects on a range of inflammatory mediators.

Four novel protease inhibitors were profiled using fluorogenic peptide substrates, before being tested for cytotoxicity by monitoring the release of lactate dehydrogenase. Downstream effects on the expression and secretion of proteinase-activated receptor 2 (PAR-2) and cytokines, IL-8 and IL-6, in CuFi cells treated with our inhibitors were investigated using qPCR and by ELISA. PAR-2 activation in a CuFi cell line was investigated through monitoring calcium mobilization.

We report NAP858, NAP1099, NAP1127 and NAP743 to be effective inhibitors of a range of CAPs. The compounds were also shown to be non-toxic to CuFi cells when treated for 24 hours when compared with vehicle alone. Initial qPCR studies indicate that treatment of CuFi cells with NAP858 and NAP1099 causes a reduction in IL-8 expression. Additionally, ELISA studies have shown that the NAP compounds are able to reduce TNFα- and LPS-stimulated IL-6 and IL-8 secretion. Calcium mobilization studies also show a reduction in PAR-2 activation after treatment with compounds.

These novel inhibitors are therefore promising follow-up compounds to QUB-TL1, presenting a potential future therapeutic option to inhibit the activation of ENaC by CAPs, as well as exerting potentially beneficial effects on downstream inflammatory signaling.

Reihill JA et al (2016) Am J Respir Crit Care Med, 194, 701-710